[1] Glycolipid Lipase
It is known that plants and microorganisms produce lipases that hydrolyze glyceroglycolipids. Enzymes derived from plants have the ability to hydrolyze mainly glycerophospholipids and glyceroglycolipids, although the ability to hydrolyze neutral fats, particularly triglycerides, is extremely low. Among microorganisms, lipases derived from actinomyces, bacteria, and molds are known. Lipases derived from actinomyces and bacteria have the property of hydrolyzing also glycerophospholipids in addition to glyceroglycolipids. It is known that lipases derived from filamentous fungi hydrolyze neutral fats, glycerophospholipids and glyceroglycolipids. As used herein, “a glyceroglycolipid lipase” refers to an enzyme that has an activity of hydrolyzing glyceroglycolipid into lysoglyceroglycolipid and fatty acid (hereinafter referred to as “glyceroglycolipid degradation activity”). A digalactosyldiacylglycerol degradation activity (hereinafter referred to as “DGDG degradation activity”) is a concept included in the glyceroglycolipid degradation activity. According to the present invention, “relative degradation activity” is defined to be a relative value of a DGDG degradation activity or a lecithin degradation activity under given pH or temperature conditions defining, as 100%, the DGDG degradation activity or the lecithin degradation activity under specific pH or temperature conditions that result in the highest activity. In addition, according to the present invention, “relative residual degradation activity” is defined to be a relative value of a DGDG degradation activity or a lecithin degradation activity under given pH or temperature conditions after treatment under the given pH or temperature conditions defining, as 100%, the DGDG degradation activity or the lecithin degradation activity under specific pH or temperature conditions that result in the highest activity after treatment under the specific pH or temperature conditions.
As to lipases derived from bacteria that hydrolyze glyceroglycolipids, for example, lipases derived from Cornebacterium efficiens, Thermobifida fusca, and the like are known (Patent Document 1).
As to lipases derived from actinomyces that hydrolyze glyceroglycolipids, for example, lipases derived from Streptomyces sp. are known (Patent Document 1).
As to lipases derived from molds that hydrolyze glyceroglycolipids, for example, lipases derived from Fusarium venenatum, Fusarium sulfureum, Fusarium culmorum, Fusarium solani, Fusarium oxysporum, Acremonium berkeleyanum, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and the like are known (Patent Documents 2-4).
[2] Glycerolipid
Glyceroglycolipids are distributed mainly among Gram-positive bacteria and chloroplasts of higher plants. Glyceroglycolipid is a compound having a sugar chain covalently bonding to the 3-position of 1,2-diacylglycerol. Galactose or the like is included as the sugar chain, and the composition ratios differ depending on the sources. For example, monogalactosyldiacylglycerol (hereinafter referred to as “MGDG”), digalactosyldiacylglycerol (hereinafter referred to as “DGDG”) and the like are included in glyceroglycolipids.
Glycerophospholipids are widely distributed among animals, plants and fungi. Glycerophospholipid is a compound having a phosphoryl base covalently bonding to the 3-position of 1,2-diacylglycerol. Choline, ethanolamine, serine, inositol, glycerol or the like is included as the base, and the composition ratios differ depending on the sources. For example, lecithin and the like are include in glycerophospholipids.
Neutral fats are also widely distributed among animals, plants and fungi. Neutral fat is a generic term for monoacylglycerol, diacylglycerol and triacylglycerol.
[3] Enzymatic Treatment of Lecithin or Glyceroglycolipid
Lecithin or glyceroglycolipid has two hydrophobic fatty acid moieties in the molecule, and is known as a lipophilic surfactant. Hydrolysis of one of them with a lipase increases the hydrophilicity, resulting in a substance having properties different from those of the lecithin or the glyceroglycolipid. Actually, since lysolecithin generated as a result of action by a phospholipase on lecithin is water soluble, and the physical properties of a food obtained using the lysolecithin as a food additive differ from those of the lecithin, its application in the food industry is examined.
Also in case of glyceroglycolipid, it is possible to produce lysoglyceroglycolipid by partially hydrolyzing it with a glyceroglycolipid lipase. Lysoglyceroglycolipid is also a substance with increased hydrophilicity. For example, lysoglyceroglycolipids include digalactosylmonoglyceride (hereinafter referred to as “DGMG”) and the like.
[4] Use of Glyceroglycolipid Lipase in Food
By using a glyceroglycolipid lipase, it is possible to produce lysoglyceroglycolipid from glyceroglycolipid in the presence of water as shown below:
wherein each of R1 and R2 represents an alkyl group, and Gal represents galactose.
If the enzyme also has a lecithin degradation activity, it is possible to simultaneously produce lysolecithin, which is lysoglycerophospholipid, from lecithin:
wherein each of R1 and R2 represents an alkyl group, and R3 represents a base such as choline, ethanolamine, glycerol, inositol or the like.
It is possible to provide a product containing a hydrophilic surfactant by leading such a reaction in a food material.
In addition, since various processes are performed on food in the range of neutral to weak acid in many cases in the food industry in order to prevent deterioration of foods, it is desirable that an enzyme preparation has a high activity in this pH range.
[5] Use of Glyceroglycolipid Lipase for Baking
Upon baking, a chemically synthesized surfactant is used in many cases for increasing the volume, improving the texture, or the like. However, baking without adding a synthetic surfactant is desirable since nature-oriented trend is growing recently. In addition, oils and fats, egg yolks, and the like are added to bread for improving the texture. However, baking without adding such an additive is also desirable since health-oriented trend is growing or the issue of allergy is serious.
It is known that flours used for baking contain neutral fats, glyceroglycolipids, glycerophospholipids, and the like (Non-patent Document 1). Among these, glyceroglycolipids and glycerophospholipids are expected to serve as surfactants, although their performance is disappointing because of their lipophilicity. Then, it would be possible to sufficiently bring out the performance by partially hydrolyzing these lipids into lysoglyceroglycolipids and lysoglycerophospholipids, which are hydrophilic surfactants. If surfactants could be supplied by components of flour itself, the use of synthetic surfactants would be unnecessary or the amount to be used could be reduced. It is known that, among flour components, the content of glyceroglycolipids is more than that of glycerophospholipids (Non-patent Document 1). Thus, it is desired in the field of baking to produce lysoglyceroglycolipids by hydrolyzing glyceroglycolipids more efficiently as compared with glycerophospholipids to increase the surface-active ability. Furthermore, it is desirable that the enzymatic activities on the produced lysoglyceroglycolipids and lysolecithins are low. In other words, it is desirable that the enzymatic activity on lysophosphatidylcholines (hereinafter referred to as “LPCs”) is low.
It has been shown that an enzyme that also hydrolyzes neutral fat in addition to glyceroglycolipid and glycerophospholipid is effective for baking (Patent Document 5). On the other hand, there is also an instance where an enzyme that hardly hydrolyzes neutral fat is more effective for baking (Patent Document 6).
Furthermore, in baking, raw materials are mixed at around pH 6 and incubated at 30-42° C. for fermentation in many cases. Thus, it is desirable that the glyceroglycolipid lipase to be used has an activity and is stable under these conditions.
[6] Problems of Known Glyceroglycolipid Lipases
Lipases derived from plants have problems with their universal use. There has been no detailed description on lipases derived from bacteria. Glyceroglycolipid lipases derived from actinomyces additionally have a lecithin degradation activity. Furthermore, many of lipases derived from filamentous fungi are derived from pathogens, thus causing some problems concerning the safety. Furthermore, the properties of the enzymes have not been described in detail. For example, it is presumed that a lipase derived from Fusarium oxysporum, which is a plant pathogen, is effective also for actual baking. However, the optimum pH of the lipase is around 9, whereas the enzymatic activity at pH 6, which is essential for baking, is only 35% or less of the enzymatic activity at the optimum pH. Regarding a lipases derived from a non-pathogen Aspergillus niger, although the enzymatic activity at pH 4.5 or 5 has been described, the activity at around pH 6, which is considered to be essential for baking, has not been shown and the effectiveness thereof is unknown.
As described above, the heretofore known glyceroglycolipid lipases have problems that: it is highly possible that they only bring insufficient effects; they have problems concerning the safety; the enzymatic reactions are inefficient; or the like. Desirable properties of glyceroglycolipid lipases include being derived from a microorganism without a problem concerning safety, having the ability to efficiently hydrolyze glyceroglycolipid and glycerophospholipid at around pH 6, being thermostable to some extent, and not hydrolyzing lysolecithin.    [Patent Document 1] WO 2006008653    [Patent Document 2] WO 2002000852    [Patent Document 3] US 2006075518    [Patent Document 4] WO 2004018660    [Patent Document 5] Japanese Patent No. 3824174    [Patent Document 6] Japanese Laid-Open Patent [Kohyo] Publication No. 2007-528732    [Non-patent Document 1] Carr N. et. al., Critical Reviews in Food Science and Nutrition, 1992, Vol. 31, p. 237-258